Bioconversion of Organic Residues for Rural Communities (UNU, 1979, 178 p.)

Integrated research on agricultural waste reclamation[edit | edit source]

Hideo Ebine

Applied Microbiology Division, National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, Tokyo, Japan

Introduction[edit | edit source]

A stable supply of feedstuffs is an absolute necessity for the sound development of the animal and fishery industries in Japan. However, the recent trend, particularly in 1972 - 73, towards world-wide constraints on foodstuff supplies has caused sharp rises in animal feed market prices. In order to cope with this, research efforts have been accelerated to improve forage production. However, our domestic feed supply is limited because most of the arable land is already cultivated, and because climatic conditions control how much can be grown. The forage supply in Japan is sufficient to meet the requirements of dairy cows and beef cattle, but hogs and poultry require a much higher concentration of protein in their feed than cattle do.

Constant supplies of soybean meal and fish meal, major sources of feed protein, will not always be ensured in view of the drastically changing patterns of crop marketing and reduced availability of fish in the world. These circumstances justify the development of alternative feed proteins, among which single-cell protein (SCP) is of prime importance. Although there are many possible substrates on which to grow SCP, production technology based on the exploitation of agricultural, forestry, and fishery waste materials is of the greatest significance, both from the standpoint of resources and environmental preservation.

In 1975, a national project to develop novel microbial protein foodstuffs from agro-waste was begun by several research institutes in the Ministry of Agriculture, Forestry, and Fisheries. These efforts are to last until 1980.

Table 1 lists the investigations being undertaken on SCP production by the National Food Research Institute, the National Forest Research Institute, and the Tokai Regional Fisheries Research Laboratory, with the assistance of the associated prefectural institutes. In addition, investigations on methods to assess the safety, feed value, and acceptability of SCP products, as well as the means to pelletize and store them, are being developed by the National Institute of Animal Health and the National Institute of Animal Industry. The most promising current potential wastes for SCP production in Japan are also listed in Table 1. These materials are available in bulk at nominal price, or even at a negative price from the food industries and forest industries.

TABLE 1. Potential Agricultural and Fishery Wastes and Institutes Where Their Utilization Is Being Studied in the Ministry of Agriculture, Forestry and Fisheries

Agro-waste Amount and property

Micro organisms

for SCP

Soybean cooking waste

380,000 570,000 tons

COD 30,000 ppm

A. oryzae NFRI*
Citrus waste 350,000 tons

Saccharomyces sp.

Candida sp.

A. oryzae

NFRI and Ehime

Prefectura Inst.

Chemical Industry

Cellulosic residue

Saw mill waste

22,000,000 tons

Rice straw

13,000,000 tons

Rice husks

2,400,000 tons

Tricoderma sp.

Candida sp.

Natl. Forest Res.



Fish processing waste

2,700,000 tons, including

160,000 tons of solids

A. tamaril

Tokai Regional

Fishery Res. Lab


* National Food Research Institute
** Kushiro Fishery Research Laboratory

Cooking waste results from the processing of miso, typical fermented soybean food in Japan. The treatment to reduce the chemical oxygen demand (COD) before discharging the waste into a river is very difficult because the COD level is so high, and because it is extremely foamy. Consequently, much research has been conducted in this area (1). The subject will be treated in more detail in a later portion of this paper.

Citrus waste is discharged mainly from the juice and canning processing of Citrus unshiu, a popular fruit in Japan. The waste, estimated to amount to approximately 350,000 tons per year, is pressed again after liming to obtain the secondary juice; this juice accounts for 50 per cent of the initial waste (2). The secondary juice, containing about ten per cent sugar, can be used as an SCP substrate. This secondary waste, which is currently dehydrated in rotating dryers for making compound feed, can also serve as a solid medium for fungus cultivation in order to raise its protein level.

A large amount of cellulosic residue is also available in Japan. The key to its use for SCP production is in developing methods to treat the materials in order to produce fermentable carbohydrate economically. Possible treatments of agro-waste materials through mechanical, chemical, and biological degradation are being intensively investigated. For example, using a cryomill, one can obtain, in a short time, a fine rice husk powder of 250 mesh or even smaller particles. The cellulosic powder, which lacks the normal fine-structure crystallinity of cellulose, is easily degrated by enzyme treatment, particularly after delignification with 1 per cent NaOH solution. Cryomill processing is promising because energy for cooling is available as a by-product from the evaporation of liquid natural gas in Japan.

The large amount of waste from the fisheries industry is an urgent problem because of water pollution. Each year, 2,700,000 tons of fish processing waste containing 6 per cent dry matter are discharged from fish meal factories. The dry matter amounts to 160,000 tons, of which 50,000 tons are utilized as feed after condensing and dehydration. The other 110,000 tons have yet to be utilized. An investigation is being undertaken to utilize this waste, which contains protein and oil, as an SCP substrate, employing a fish oil-assimilating fungus, Aspergiflus tamarii, isolated from tamari-miso, which in itself contains a comparatively high level of oil.

Special considerations are paid to the safety of both the ingredients and the microorganisms selected for processing. In screening tests, suitable micro-organisms have been isolated from the traditional fermented foods such as miso, shoyu, and sake in Japan. For example, as shown in Table 2. Aspergillus oryzae, A. sojae, and A. tamarii are widely employed in the fermented food industries. The total amount of koji, the fermented products of these fungi, is approximately 900,000 tons per year. The mycelium content of koli, as determined by Arima (3), differs widely depending upon the ingredients and culture conditions, including duration, temperature, relative humidity, level of oxygen and carbon dioxide in the fermenting facility, and mechanical agitation of the materials. The total amount of mycelium in koji is calculated to be approximately 73,000 tons, which have been traditionally eaten as part of miso, shoyu, sake, and other fermented foods made with koji. This fact is very important for the acceptance of novel microbial protein either as a food or feed prepared with Aspergillus oryzae or its related strains.

TABLE 2. Amount of Mycelium in Aspergillus for Several Kinds of Koji Food Processing in Japan


in koji





Mycelium in koji (%) Amount (Tons)
Sake Rice 120,000 132,000 1.2 1,585
Mirin Rice 3,000 3,300 2.6 85
Miso Rice 100,000 106,000 5.0 5,300
Barley 30,000 31,800 5.0 1,590
Soybeans 26,000 24,310 16.6 3.560
Shoyu Soybeans 190,000 350 000 16.6 58,930
Wheat 190,000
Miscellaneous Rice 30,000 31,800 5.0 1,590
Total 689,000 679,210 72,640

Production of yeast from soybean cooking waste at miso factories[edit | edit source]

Because soybean cooking waste has such a high COD value, it is one of the most difficult of all food industry wastes to treat in Japan. Consequently, there are many devices to treat soybean cooking waste via mechanical, chemical, or biological methods.

In 1970, an industrial co-operative was formed to treat this waste. It was established by nine members from a miso factory at Maruko, Nagano Prefecture (4). This is the sole factory in Japan where SCP is being made from agro-industrial waste, except for factories that make torula yeast from spent sulphite liquor.

As shown in Figure 1, soybean cooking waste is sent to the factory by tank trucks or pipelines directly from the miso factory cookers. After the pH value is adjusted to 4.0 in a serving tank, the waste is transferred into a Waldhof continuous fermenter of 15 kl capacity. In the fermenter, Torulopsis xylinus is cultivated, with an anti-foaming agent, at 30°Cat a dilution rate of 0.3 - 0.4 hr(-1) without any other nutrient supplements. Maximum production of dehydrated yeast is 800 kg when 80 tons of waste are supplied and COD is reduced by 70 - 75 per cent. For purpose of lowering production costs, the yeast milk, after washing and heating at 80°C for 30 min., is often delivered to neighborhood farms.

Figure. 1. Flow Diagram of Production of Fodder Yeast from Soybean Cooking Waste

At present, this method has problems that must be solved: (i) the 70 - 75 per cent COD reduction rate should be raised still further; (ii) microbial contamination, originating mainly during transportation of the waste in tank trucks, must be eliminated; and (iii) the process, although small in scale, requires trained technologists to conduct it properly, resulting in higher costs to the consumer.

Application of soy waste as koji substrate for rice miso manufacturing (5, 6)[edit | edit source]

Milled rice is used for making rice koji, which supplies the necessary enzymes for the fermentation of rice miso. However, during the 48-hour period of koji preparation, approximately 10 per cent of the solids are consumed by the fungi. Soybeans also lose approximately 10 per cent of their solids, though the rate varies widely, depending on soaking and cooking methods. This investigation, there fore, was designed not only to determine the best way to manufacture rice miso, but also to explore the utilization of soybean cooking waste as a substrate for cultivating the fungus, Aspergillus. Soybean waste contains all the nutrients required by Aspergillus and also promotes the fermentation of miso (7). If successful, the results should yield the following advantages:

1) reduction of the COD value of soybean cooking waste by 80 per cent or more;

2) an up-grading of the biomass to food level, and

3) lowering the amount of rice koji needed, thereby eliminating the necessity of using so much expensive rice as an ingredient.

After screening tests employing 28 strains of fungi, including Aspergillus sp., Rhizopus sp., Penicillium sp., and Paecilomyces sp., we selected Aspergillus oryzae FRI-23 for the experiment. It was isolated from commercial tanekoji (fermented brown rice) and proved to be free of mycotoxins.

Soybean cooking waste with a COD of 20,000 ppm gave the highest rate of growth and the best reduction of COD, as shown in Figure 2. Cultivation was conducted at 30°C under conditions of 1 vvm at a stirring rate of 400 rpm for 24 hours. At that time, the proteolytic enzyme activity attained a peak. At this stage, except for amylase, most of the proteolytic enzymes, particularly polypeptidases, were found to remain in the cells. As shown in Table 3, except for acid proteinase and amylase, the activity of essential enzyme produced in the cooking waste from 1,000 kg soy beans was higher than that in rice koji made from 700 kg of rice. This fact suggests the possibility of replacing the koji from rice with the mycelium grown in the waste when miso is made from 1,000 kg of soybeans, 700 kg of rice, and 430 kg of salt, or the same ratio of these ingredients.

TABLE 3. Enzyme Activity of Mycelium Made from Soybean Cooking Waste and Rice Koji


Enzyme activity

Mycelium (x 1,000)

Rice koji (x 1,000)
Proteinase (pH 3) 23,200 42,000
(pH 6) 36,000 35,280
(pH 7.5) 17,200 15,960
Acid carboxypeptidase 144 84
Leucine aminopeptidase 188 59
Amylase 1.2 1,176

Figure. 2. Flow Diagram of Miso Fermentation with Supplementation of Mycelium Grown in Cooking Waste

The mycelium was forced through a filter cloth and pressed to an 80.5 per cent moisture level. After chopping and grinding, the mycelium was mixed with green miso, prepared by mixing cooked soybeans and salted rice koji with an inoculum that included salt-resistant lactic acid bacteria and yeast. After fermentation, this new type of miso, containing two to five per cent of wet, living mycelium, showed a more advanced fermentation and degree of ripening than did the conventional miso

As illustrated in Table 4, the amounts of amino acids liberated from the protein of the mycelium-containing miso

TABLE 4. Effect of Mycelia on the Liberation of Free Amino Acids and Amides of Miso (mg/100 g)


0 Days*

67 Days

2% Mycelia

67 Days

5% Mycelia

67 Days

Asp-NH 60 104 176 229
Glu-NH2 103 271 360 428
Lysine 73 188 220 248
Histidine 17 36 42 61
Arginine 133 277 244 221
Asparagine 27 130 154 170
Threonine 30 119 132 188
Serine 41 157 186 232
Glutamine 62 249 311 381
Proline 23 101 109 115
Glycine 12 58 73 92
Alanine 34 125 155 188
Cystine 28 78 71 70
Valine 25 124 149 167
Methionine 17 42 63 70
Isoleucine 18 110 132 153
Leucine 40 223 260 289
Tyrosine 23 143 144 174
Phenylalanine 38 197 189 228

* Immediately after mixing rice koji, soy cooking waste, salt, and water for fermentation.

Mycelium enzyme represents the total amount of enzyme in the mycelium grown in the cooking waste from 1,000 kg of soybeans. Rice koji enzyme represents the total amount of enzyme in the rice koji made from 700 kg of rice were greater than those found in conventional miso. The soy waste mycelium also accelerated the growth and fermentation of the micro-organisms added as starters, thus playing a very important role in the formation of the attractive flavours found in ripened miso.

The amino acid patterns of the mycelium were similar to those in biomass grown on acetate. It is of interest that the content of nucleic acids, including RNA and DNA in mycelium, was 4 per cent on a dry weight basis (Table 5).

TABLE 5. Amino Acid, RNA and DNA Composition of Soybean

Cooking Waste and Mycelia of A. oryzae FRI-23


(g 100/ml, 100 g dry mycelia)

Amino acids
Asparagine 0.071 3.3
Threonine 0.019 1.6
Serine 0.019 1.7
Glutamine 0.151 4.7
Proline 0.032 1.3
Glycine 0.024 1.5
Alanine 0.020 2.0
Cystine - -
Valine 0.018 1.9
Methionine (0.007) (0.6)
Isoleucine 0.014 1.4
Leucine 0.022 2.3
Tyrosine 0.014 1.2
Phenylalanine 0.019 5.5
Lysine 0.039 3.0
Histidine 0.016 1.0
Arginine 0.058 1.9
Tryptophan - -
RNA - 3.5
DNA - 0.5
Crude protein*** 0. 11 40. 0

* Soybean cooking waste (COD 20,000 ppm)
** Shaking culture at 30°C for 72 hours
*** T.N. x6.25

Conclusion[edit | edit source]

The utilization of the wastes from the food industries is beset with many problems, among which economic feasibility is of prime importance, particularly for comparatively smallscale factories. As an example of one solution for coping with these problems, the use of soybean cooking waste as a substrate for koji-mould cultivation was investigated. The biomass obtained contributed not only protein and other nutrients, but also enhanced enzyme activity for the fermentation of miso, thus providing an apparent economic advantage.

References[edit | edit source]

1. Food Agency, Ministry of Agriculture, Forestry, and Fisheries, Japan, Standards for Management of Pollution in Miso Factories, 1977.

2. R. Hendrickson and J.W. Kesterson, "Citrus Molasses," University of Florida Agric. Expt. Stat Bulk 677 (1964).

3. K. Arima and T. Uozumi, "A New Method for Estimation of the Mycelium Weight in Koji," Agric. Biol. Chem. 31: 1-19 (1967)

4. T. Mochizuki, "Utilization of Soybean Cooking Waste as a Medium for Yeast Production," Misono Kagaku to Gijutsu 201: 2 (1970).

5. M. Yanagimoto, H. Saito, and H. Ebine, "Cultivation of Aspergillus oryzae in Soybean Cooking Waste," J. Soc. Brew. Japan 70:424 (1975).

6. S. Nikuni, H. Ito, H. Takagi, and H. Ebine, "Utilization of Soybean Cooking Drain," Sbokuhin Kogyo Gakkasishi 26 (in press).

7. S. Hayashida, N. Minamisato, T. Tei, and M. Hongo, "The High Concentration of Alcohol-Producing Factor in Koji," Nogeikagakukaishi 48 : 529 (1974).

Discussion summary[edit | edit source]

Questions concerning the extent to which SCP production on secondary juice from citrus peel is applied in Japan, and about the economics thereof, cannot be adequately answered until the project is completed in 1980.

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Discussion[View | Edit]

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